, Volume 221, Issue 3, pp 424–436 | Cite as

Arabidopsis emb175 and other ppr knockout mutants reveal essential roles for pentatricopeptide repeat (PPR) proteins in plant embryogenesis

  • Daniel A. Cushing
  • Nancy R. Forsthoefel
  • Daniel R. Gestaut
  • Daniel M. VernonEmail author
Original Article


Pentatricopeptide repeat proteins (PPRPs) constitute one of the largest superfamilies in plants, with more than 440 identified in the Arabidopsis thaliana (L.) Heynh genome. While some PPRPs are known to take part in organelle gene expression, little is known about the broader biological contexts of PPRP gene function. Here, using developmental- and reverse-genetic approaches, we demonstrate that a number of PPRPs are essential early in plant development. We have characterized the Arabidopsis embryo-defective175 mutant and identified the EMB175 gene. Emb175 consistently displays aberrant cell organization and undergoes morphological arrest before the globular-heart transition. The emb175 mutation disrupts an intronless open reading frame encoding a predicted chloroplast-localized PPR protein— the first to be rigorously associated with an early embryo-lethal phenotype. To determine if other PPRP genes act in embryogenesis, we searched Arabidopsis insertion mutant collections for pprp knockout alleles, and identified 29 mutants representing 11 loci potentially associated with embryo-defective phenotypes. We assessed gene structures, T-DNA insertion position, and allelism for these loci and were able to firmly establish essential functions for six PPRP genes in addition to EMB175. Interestingly, Nomarski DIC microscopy revealed diverse embryonic defects in these lines, ranging from early lethality to dramatic late-stage morphological defects such as enlarged shoot apices and stunted cotyledons. Together, emb175 and these pprp knockout mutants establish essential roles for PPRPs in embryogenesis, thus broadening the known organismal context for PPRP gene function. The diversity of emb–pprp knockout phenotypes indicates that mutation of different PPRPs can, directly or indirectly, have distinct impacts on embryo morphogenesis.


Arabidopsis Embryogenesis Knockout mutants Morphogenesis Plant development PPR motif 





Differential interference contrast


Inverse polymerase chain reaction


Open reading frame


Pentatricopeptide repeat


Pentatricopeptide repeat protein




Untranslated region



We are grateful for support from the USDA (02-35304-12304 to DMV) and the Murdock Charitable Trust. Emb175 characterization was initiated with support from NSF-9604344 to DMV. The W.M. Keck Foundation provided support for digital imaging equipment. We thank Dr. David Meinke (Oklahoma State University) for emb175 complementation crosses with emb1899-1 and −2 when those mutants were identified in the course of this work. cDNAs and mutant seed stocks were provided by the ABRC. We thank Tovi M. Anderson and John E. Houghland for assistance.


  1. Albert S, Despres B, Guilleminot J, Bechtold N, Pelletier G, Delseny M, Devic M (1999) The EMB506 gene encodes a novel ankyrin repeat containing protein that is essential for the normal development of Arabidopsis embryos. Plant J 17:169–179CrossRefGoogle Scholar
  2. Alonso JM et al (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301:653–657CrossRefPubMedGoogle Scholar
  3. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402PubMedGoogle Scholar
  4. Apuya NR, Yadegari R, Fischer RL, Harada JJ, Goldberg RB (2002) Raspberry3 gene encodes a novel protein important for embryo development. Plant Physiol 129:1–15CrossRefGoogle Scholar
  5. Aubourg S, Boudet N, Kreis M, Lecharny A (2000) In Arabidopsis thaliana, 1% of the genome codes for a novel protein family unique to plants. Plant Mol Biol 42:603–613CrossRefGoogle Scholar
  6. Barkan A, Walker M, Nolasco M, Johnson D (1994) A nuclear mutation in maize blocks the processing and translation of several chloroplast mRNAs and provides evidence for the differential translation of alternative mRNA forms. EMBO J 13:3170–3181Google Scholar
  7. Bateman A, Coin L, Durbin R, Finn RD, Hollich V, Griffiths-Jones S, Khanna A, Marshall M, Moxon S, Sonnhammer EL, Studholme DJ, Yeats C, Eddy SR (2004) The Pfam protein families database. Nucleic Acids Res 32:D138-D141CrossRefGoogle Scholar
  8. Bentolila S, Alfonso AA, Hanson MR (2002) A pentatricopeptide repeat-containing gene restores fertility to cytoplasmic male-sterile plants. Proc Natl Acad Sci USA 99:10887–10892CrossRefPubMedGoogle Scholar
  9. Castle LA, Errampalli D, Atherton T, Franzmann, Yoon E, Meinke DW (1993) Genetic and molecular characterization of embryonic mutants identified following seed transformation in Arabidopsis. Mol Gen Genet 241:504–514PubMedGoogle Scholar
  10. Coffin JW, Dhillon R, Ritzel RG, Nargang FE (1997) The Neurospora crassa cya-5 nuclear gene encodes a protein with a region of homology to the Saccharomyces cerevisiae PET309 protein and is required in a post-transcriptional step for the expression of the mitochondrially encoded COXI protein. Curr Genet 32:273–280CrossRefGoogle Scholar
  11. Despres B, Delseny M, Devic M (2001) Partial complementation of embryo defective mutations: a general strategy to elucidate gene function. Plant J 27:149–159CrossRefGoogle Scholar
  12. Emanuelsson O, Nielsen H, Brunak S, von Heijne G (2000) Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol 300:1005–1016CrossRefPubMedGoogle Scholar
  13. Errampalli D, Patton D, Castle L, Mickelson L, Hansen K, Schnall J, Feldmann K, Meinke DW (1991) Embryo lethals and T-DNA insertion mutagenesis in Arabidopsis. Plant Cell 3:149–157CrossRefGoogle Scholar
  14. Fisk DG, Walker MB, Barkan A (1999) Molecular cloning of the maize gene crp1 reveals similarity between regulators of mitochondrial and chloroplast gene expression. EMBO J 18:2621–2630CrossRefGoogle Scholar
  15. Forsthoefel NR, Wu Y, Shulz B, Bennett, MJ, Feldmann KA (1992) T-DNA insertion mutagenesis in Arabidopsis: prospects and perspectives. Aust J Plant Physiol 19:353–366Google Scholar
  16. Franzmann L, Yoon E, Meinke DW (1995) Saturating the genetic map of Arabidopsis thaliana with embryonic mutations. Plant J 7:341–350CrossRefGoogle Scholar
  17. Goldberg RB, de Paiva G, Yadegari R (1994) Plant embryogenesis: zygote to seed. Science 266:605–614Google Scholar
  18. Hashimoto M, Endo T, Peltier G, Tasaka M, Shikanai T (2003) A nucleus-encoded factor, CRR2, is essential for the expression of choloplast ndhB in Arabidopsis. Plant J 36:541–549CrossRefGoogle Scholar
  19. Ikeda TM, Gray MW (1999) Characterization of a DNA-binding protein implicated in transcription in wheat mitochondria. Mol Cell Biol 19:8113–8122PubMedGoogle Scholar
  20. Arabidopsis Genome Initiative (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408:796–826CrossRefPubMedGoogle Scholar
  21. Kazama T, Toriyama K (2003) A pentatricopeptide repeat-containing gene that promotes the processing of aberrant atp6 RNA of cytoplasmic male-sterile rice. FEBS Lett 544:99–102CrossRefPubMedGoogle Scholar
  22. Koc EC, Spremulli LL (2003) RNA-binding proteins of mammalian mitochondria. Mitochondrion 2:277–291CrossRefGoogle Scholar
  23. Komori T, Ohta S, Murai N, Takakura Y, Kuraya Y, Suzuki S, Hiei Y, Imaseki H, Nitta N (2004) Map-based cloning of a fertility restorer gene, Rf-1, in rice. Plant J 37:315–325CrossRefPubMedGoogle Scholar
  24. Liu L, McKeehan WL (2002) Sequence analysis of LRPPRC and its SEC1 domain interaction partners suggests roles in cytoskeletal organization, vesicular trafficking, nucleocytosolic shuttling and chromosome activity. Genomics 79:124–136CrossRefGoogle Scholar
  25. Lurin C et al (2004) Genome-wide analysis of Arabidopsis pentatricopeptide repear proteins reveals their essential role in organelle biogenesis. Plant Cell 16:2089–2103CrossRefGoogle Scholar
  26. Mancebo R, Zhou X, Shillinglaw W, Henzel W, Macdonald PM (2001) BSF binds specifically to the bicoid mRNA 3′ untranslated region and contributes to stabilization of bicoid mRNA. Mol Cell Biol 21:3462–3471CrossRefGoogle Scholar
  27. Manthey GM, McEwenJE (1995) The product of the nuclear gene PET309 is required for translation of mature mRNA and stability or production of intron-containing RNAs derived from the mitochondrial COX1 locus of Saccharomyces cerevisiae. EMBO J 14:4031–4043Google Scholar
  28. McElver J, Tzafrir I, Aux G, Rogers R, Ashby C, Smith K, Thomas C, Schetter A, Zhou Q, Cushman MA, Tossberg J, Nickle T, Leven JZ, Law M, Meinke D, Patton D (2001) Insertional mutagenesis of genes required for seed development in Arabidopsis thaliana. Genetics 159:1751–1763Google Scholar
  29. Meierhoff K, Felder S, Nakamura T, Bechtold N, Schuster G (2003) HCF152, and Arabidopsis RNA binindg pentatricopeptide repeat protein involved in the processing of Chloroplast psbB-psbT-H-petB-D RNAs. Plant Cell 14:1480–1495CrossRefGoogle Scholar
  30. Meinke DW (1995) Molecular genetics of plant embryogenesis. Annu Rev Plant Physiol Plant Mol Biol 46:369–394CrossRefGoogle Scholar
  31. Mulder NJ et al (2003) The InterPro Database, 2003 brings increased coverage and new features. Nucleic Acids Res 31:315–318CrossRefPubMedGoogle Scholar
  32. Sambrook J, Fritsch EF, Maniatis, T (1989) Molecular Cloning: a laboratory manu al . Cold Spring Harbor, Cold Spring HarborGoogle Scholar
  33. Schwartz BW, Yeung EC, Meinke DW (1994) Disruption of morphogenesis and transformation of the suspensor in abnormal suspensor mutants of Arabidopsis. Development 120:3235–3245Google Scholar
  34. Schwartz BW, Vernon DM, Meinke DW (1997) Development of the suspensor: differentiation, communication, and programmed cell death during plant embryogenesis. In: Larkins BA, Vasil IK (eds) Cellular and molecular biology of plant seed development. Kluwer, Dordrecht, pp 73–115Google Scholar
  35. Small ID, Peeters N (2000) The PPR motif: a TRP-related motif prevalent in plant organeller proteins. Trends Biochem Sci 2:46–47Google Scholar
  36. Small ID, Peeters N, Legeai F, Lurin C (2004) Predotar: a tool for rapidly screening proteomes for N-terminal targeting sequences. Proteomics 4:1581–1590CrossRefGoogle Scholar
  37. Tax FE, Vernon DM (2001) T-DNA associated duplication/translocations in Arabidopsis: implications for mutant analysis and functional genomics. Plant Physiol 126:1527–1538CrossRefPubMedGoogle Scholar
  38. Tsuchiya N, Fukuda H, Sugimura T, Nagao M, Nakagama H (2002) LRP130, a protein containing nine pentatricopeptide repeat motifs interacts with a single-stranded cytosine-rich sequence of mouse minisatellite Pc-1. Eur J Biochem 269:2927–2933CrossRefGoogle Scholar
  39. Tzafrir I, Pena-Muralla R, Dicherman A, Berg M, Rogers R, Hutchens S, Sweeney TC, McElver J, Aux G, Patton D, Meinke D (2004) Identification of genes required for embryo development in Arabidopsis. Plant Physiol 135:1206–1220CrossRefGoogle Scholar
  40. Vernon DM, Meinke DW (1994) Embryogenic transformation of the suspensor in twin, a polyembryonic mutant of Arabidopsis thaliana. Dev Biol 165:566–573CrossRefGoogle Scholar
  41. Vernon DM, Meinke DW (1995) Late embryo-defective mutants of Arabidopsis. Dev Genet 16:311–320Google Scholar
  42. West MA, Harada JJ (1993) Embryogenesis in higher plants: an overview. Plant Cell 5:1361–1369CrossRefGoogle Scholar
  43. Williams PM, Barkan A (2003) A chloroplast-localized PPR protein required for plastid ribosome accumulation. Plant J 36:675–686CrossRefGoogle Scholar
  44. Yadegari R, De Paiva G, Laux T, Kalunow AM, Apuya N, Zimmerman JL, Fischer B, Harada JJ, Goldberg RB (1994) Cell differentiation and morphogenesis are uncoupled in Arabidopsis raspberry embryos. Plant Cell 6:1713–1729CrossRefGoogle Scholar
  45. Yamazaki H, Tasaka M, Shikanai T (2004) PPR motifs of the nucleus-encoded factor PGR3 function in selective and distinct steps of chloroplast gene expression in Arabidopsis. Plant J 38:152–163CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Daniel A. Cushing
    • 1
  • Nancy R. Forsthoefel
    • 1
  • Daniel R. Gestaut
    • 1
  • Daniel M. Vernon
    • 1
    Email author
  1. 1.Department of Biology, and Program in Biochemistry, Biophysics and Molecular BiologyWhitman CollegeWalla WallaUSA

Personalised recommendations